Method for preparing inactivated vaccine of encephalitis B used for human being

A technology of Japanese encephalitis and inactivated vaccines, which is applied in the direction of resisting vector-borne diseases, virus antigen components, antiviral agents, etc., can solve the problem of inability to use human diploid cells, and is beneficial to quality control and preparation The method is scientific and reasonable, and the effect of high output

Inactive Publication Date: 2007-05-23
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these commonly used strains such as P3, SA 14-14-2, etc. are not sensitive to human diploid cells. Therefore, human diploid cells have not been able to be used in the production of JE vaccines for a long time.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1——carry out virus identification to DL4 strain

[0026] It was confirmed to be JE virus by filtration test, neutralization test with JE specific immune serum, cross HI and IFAT test of monoclonal antibody. The results of electron microscope and pathological examination showed that DL4 virus could reproduce in the brain tissue of mice and cause the degeneration and necrosis of nerve cells, which was consistent with the neurotropism of JE virus. The neurotropic virulence of this strain was detected by JE-sensitive animal experiments. After being inoculated into 3-day-old suckling mice, after an incubation period of 8 days, regular morbidity and death occurred, and the onset showed unsteady walking and paralysis of hind limbs. After the onset of DL4-infected suckling mice, the results of histopathological examination of various organs were as follows: mild subpia mater hyperemia, focal hemorrhage in the brain parenchyma, general swelling and denaturation of ne...

Embodiment 2

[0029] Embodiment 2——to the subsection detection of vaccine and animal test

[0030] The virus harvest liquid, the virus stock solution and the vaccine stock solution are all clear liquids without foreign matter and precipitation, no bacterial pollution, no mycoplasma pollution, and no abnormal toxins, pH 7.2-8.0.

[0031] The virus harvest liquid was serially diluted 10 times, and 6 dilutions were taken. In each dilution, 4 mice with a body weight of 7-9g were inoculated into the brain, each with 0.03ml, and observed daily. Mice that died within 3 days were not counted. 14 days, the titer is not lower than 5.5lg LD 50 / ml.

[0032]After SDS-PAGE electrophoresis of a small amount of virus stock solution, Coomassie brilliant blue staining and Western detection of JE virus E protein antibody showed a single band with an apparent molecular weight of 53kDa.

[0033] KMB17 cells were inoculated with the virus stock solution, and there was no cytopathic change and no virus plaque ...

Embodiment 3

[0035] Embodiment 3—animal test for safety detection

[0036] Intraperitoneally immunize 10 mice with a body weight of 12-14 g with the stock solution of the vaccine, each with 0.5 ml, and immunize twice with an interval of 7 days. Blood was collected on the 7th day after the second immunization, the serum was separated, mixed with the same amount of serum in the same group, and inactivated at 56 degrees for 30 minutes. Dilute the serum and mix it with the diluted virus (about 200PFU / 0.4ml) in equal amounts. At the same time, mix the diluted virus with the normal mouse serum in equal amounts. As a virus control, put it in 37°C water for 90 minutes and inoculate BHK21 cells in a 6-well plate. 0.4ml per well, cultured at 37°C for 90 minutes, added a medium cover containing methylcellulose, cultured in a 5% carbon dioxide incubator at 37°C for 5 days, stained, and counted plaques showed that the vaccine solution C induced The antiserum was able to completely neutralize the virus...

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Abstract

The invention relates to an encephalitis-B inactivated vaccine, and relative production, wherein it uses human diploid cell KMB17 to cultivate encephalitis B virus DL4, collects clear virus liquid, inactivates via 1:2000 formaldehyde, dialyses, concentrates, purifies via Sepharose 4FF post, and adsorbs via aluminum hydroxide. The inventive vaccine has immunogen, with high safety, while it can prevent encephalitis B.

Description

technical field [0001] The invention belongs to the technical field of human preventive medicine, and in particular relates to an inactivated Japanese encephalitis vaccine for humans and a preparation method of the vaccine. Background technique [0002] Epidemic Japanese encephalitis, referred to as Japanese encephalitis, is an acute infectious disease of the central nervous system caused by the Japanese encephalitis virus, transmitted by mosquitoes, and zoonotic. The pathogen of Japanese encephalitis is Japanese encephalitis virus, which belongs to the Flavivirus genus of the Flaviviridae family. It is a neurotropic and RNA virus with a spherical shape and a diameter of 40nm. It has strong resistance to low temperature and dryness. JE is a regional infectious disease in Southeast Asia and the Western Pacific region, and is closely related to the local climate conditions and the vicious activities of vector mosquitoes. The prevalence of Japanese encephalitis in China has ob...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/14
CPCY02A50/30
Inventor 李琦涵刘龙丁王丽春廖芸寸韡梁燕杨子江董承红王晶晶赵红玲纳锐雄姜莉施海晶王蕾
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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